Thomas Alexander Janert

ABSTRACT: The success of targeted therapies for hematological malignancies has heralded their potential as both salvage treatment and early treatment lines, reducing the need for high-dose, intensive, and often toxic chemotherapeutic regimens. For young patients with classic Hodgkin lymphoma (cHL), immunotherapies provide the possibility to lessen long-term, treatment-related toxicities. However, suitable therapeutic targets are lacking. By integrating single-cell dissection of the tumor landscape and an in-depth, single-cell-based off-tumor antigen prediction, we identify CD86 as a promising therapeutic target in cHL. CD86 is highly expressed on Hodgkin and Reed-Sternberg cancer cells and cHL-specific tumor-associated macrophages. We reveal CD86-CTLA-4 as a key suppressive pathway in cHL, driving T-cell exhaustion. Cellular therapies targeting CD86 had extraordinary efficacy in vitro and in vivo and were safe in immunocompetent mouse models without compromising bacterial host defense in sepsis models. Our results prove the potential value of anti-CD86 immunotherapies for treating cHL.

The success of targeted immunotherapies for hematological malignancies has heralded their potential as salvage therapies as well as in earlier treatment lines (Cappell & Kochenderfer, 2023). While conventional chemotherapy-based treatments can achieve long-term survival in up to 90 % of treated patients with classic Hodgkin lymphoma (cHL), these therapies are associated with treatment-related comorbidities, calling for more tailored and specific approaches (Schaapveld et al., 2015; Shanbhag & Ambinder, 2018). While targeted treatments, especially immunotherapies are taking oncology by storm, the utility in cHL is so far limited to CD30 and PD-1-targeting strategies and there is a clear lack of drugable relevant target structures in this disease. This can be partly attributed to technical difficulties of analyzing the malignant Hodgkin-Reed-Sternberg (HRS) cells specifically. Capitalizing on our previous work using large scale data mining to inform target discovery, we hypothesized that combining different analytical methods with large single-cell RNA-Sequencing (scRNA-Seq) datasets would permit selective target definition with functional relevance to the disease and thereby allow the development of novel immunotherapeutic strategies. Leveraging microarray profiles of laser-dissected HRS cells and a scRNA-Seq cohort of cHL patients (total of n = 44 primary samples; n = 34 cHL samples; n = 10 RLN (reactive lymph node) control samples), we screened for novel target antigens highly expressed on HRS cells with functional relevance in the tumor microenvironement (TME) of cHL. Unbiased in silico analyses revealed CD80, CD86 and PD-L1 as most suitable candidate target antigens with CD86 showing the highest expression on HRS cells. ScRNA-Seq analyses unveiled a shift of the CD80-CD86-CTLA-4-CD28 towards the immunosuppressive CTLA-4 axis in the TME of cHL compared to RLN controls. In advanced cell culture models, including iPSC-derived organoid models, blockage of CD86 lead to the decreased expression of PD-1 and CTLA-4 and an overall reversal of the exhaustive phenotype of cHL-associated T cells. High protein expression of CD86 on HRS cells and in the TME (cHL-infiltrating tumor-associated macrophages (cHL-TAM), B cells) was confirmed in different validation cohorts including relapsed and refractory cHL (r/r cHL) patients by conventional immunohistochemistry and multiplexed immunofluorescence (n = 34 cHL patients). Following target identification, CAR T cells redirected against CD86 were developed and the functionality of these CAR T cells was investigated in preclinical models both in vitro and in vivo. Anti-CD86 CAR T cells effectively deplete cHL-TAM and are highly effective in various in vitro and in vivo models of cHL, including models of CD30-negative disease. Given the fundamental role of the CD80-CD86-CTLA-4-CD28 axis in the generation of the adaptive immune response, detailed toxicity assessments were carried out leveraging murine surrogate anti-CD86 CAR T cells, with similar binding and activation thresholds as their human counterpart. These anti-mCD86 CAR T cells did not cause toxicities in lymphodepleted, immunocompetent mice. In addition, the impact of anti-CD86-directed immunotherapies (e.g. anti-CD86-blocking antibodies, anti-mCD86 CAR T cells) on bacterial host defense and formation of antigen-specific adaptive immunity was investigated in syngeic mouse models. Anti-CD86 immunotherapy did not lead to enhanced bacteremia in a model of gram-negative sepsis, while preclinical vaccination models revealed a mildy reduced formation of antigen-specific T cell development in mice. In summary, we provide a framework for unbiased, multi-dimensional target screening and highlight the functional relevance of the immunosuppressive CD86-CTLA-4 axis in cHL. CD86-directed immunotherapy could reverse the exhaustive phenotype of cHL-associated T cells, while demonstrating strong treatment efficacy in xenograft mouse models. Importantly, elaborate toxicity assessments of anti-CD86-targeted immunotherapies utilizing syngenic mouse models did not reveal measureable toxicity in mice. Overall, our data emphasizes the vast translational potential of CD86-targeted immunotherapies in cHL and provide a strong rationale for further clinical investigations.

<h3>Background</h3> The potential of T-cell engaging bispecific antibodies (TCEs) is illustrated by the success of targeting CD19 (Blinatumomab) and CD20 (Epcoritamab, Glofitamab, Mosuenetuzumab) in relapsed and refractory B-cell non-Hodgkin lymphoma. However, in AML, TCEs targeting CD33 (JNJ-67571244, AMG330) and CD123 (Vibecotamab) have shown low efficacy and high toxicity. This is paralleled by preclinical studies addressing these targets evidencing on-target off-tumor toxicity against hematopoietic stem and progenitor cells (HSPCs). To address the need for safer, effective immunotherapeutic targets in AML, we previously identified the colony-stimulating factor 1 receptor (CSF1R) using an unbiased single-cell RNA sequencing approach. To further evaluate its potential efficacy and safety profile, we developed a CSF1R-targeting TCE structurally based on the CrossMAb^® technology in the 2+1 format shared with the FDA-approved CD20-targeting TCE Glofitamab. <h3>Materials and Methods</h3> In co-culture experiments with T cells or peripheral blood mononuclear cells and human AML cell lines (Mv4-11, THP-1, OCI-AML3, PL-21) as well as primary AML blasts, the binding of CSF1R-TCE was characterized using flow cytometry, while luciferase bioluminescence and live cell imaging readouts investigated the killing capacity.The on-target off-tumor toxicity against HSPCs <i>in vitro</i> was analyzed using multicolor flow cytometry or classical colony-forming unit assays. On-target-off-tumor-toxicity <i>in vivo</i> was analyzed in CD34⁺ cord blood (CB)-stem cell-humanized as well as using fully syngeneic, immunocompetent mouse models. Xenograft-derived AML cell line models were used to evaluate efficacy of CSF1R-TCE in humans. <h3>Results</h3> First, we confirmed dose-dependent binding of our CSF1R-TCE and specific lysis of AML cell lines (effector-to-target (E:T) ratio 1:2, p &lt; 0.0001 for both Mv4-11 and THP-1) and primary AML blasts (E:T 1:1, p &lt; 0.0001). The CSF1R-TCE did not cause relevant HSPC lysis <i>in vitro</i>, whereas the CD33-TCE led to a near complete depletion of HSPCs (E:T 2:1, p &lt; 0.0001). These results were confirmed in a CD34⁺ CB-stem cell-humanized mouse model, in which CSF1R-TCE showed less evidence of cytokine release and minimal HSPC count reduction compared to CD33-TCE. Similarly, in fully syngeneic mouse models, we observed no signs of on-target-off-tumor toxicity in mice treated with a CSF1R-TCE targeting the murine receptor. In the Mv4-11 AML xenograft model, the CSF1R-TCE reduced tumor outgrowth and progression compared to a control TCE (p &lt; 0.0001). <h3>Conclusions</h3> We demonstrated the safety and efficacy of our CSF1R-TCE <i>in vitro</i> and <i>in vivo</i>. Consistent with our single-cell RNA sequencing-based target analysis, the CSF1R-TCE exhibited a superior safety profile than the CD33-TCE while maintaining anti-tumor activity. CSF1R-targeting TCEs may represent a novel immunotherapeutic approach for AML with high translative potential. <b>A. Gottschlich:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Tabby Therapeutics, Nanogami. <b>T. Janert:</b> None. <b>G. Hoffmann:</b> None. <b>J. Sam:</b> A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. <b>S. Gebhardt:</b> A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. <b>L. Rohrbacher:</b> None. <b>S. Nandi:</b> None. <b>E. Carlini:</b> None. <b>T. Herold:</b> D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; Jazz Pharmaceuticals, Servier Deutschland. <b>M. von Bergwelt-Balidon:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TABBY, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, KITE/Gilead Mologen, Miltenyi, MSD Sharp + Dohme, Novartis, Priothera, Roche. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; TABBY, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, KITE/Gilead Mologen, Miltenyi, MSD Sharp + Dohme, Novartis, Priothera, Roche. F. Consultant/Advisory Board; Significant; TABBY, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, KITE/Gilead Mologen, Miltenyi, MSD Sharp + Dohme, Novartis, Priothera, Roche. <b>S. Endres:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Arcus Bioscience, Plectonic GmbH, TCR2 Inc, Catalym GmbH. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2 Inc, Carina Biotech. <b>M. Subklewe:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Amgen, BMS/Celgene, Gilead/Kite, Janssen, Miltenyi Biotec, Molecular Partners, Novartis, Roche, Seagen, Takeda. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; AstraZeneca, BMS, Gilead/Kite, GSK, Janssen, LAWG, Novartis, Pfizer, Roche, Springer Healthcare, AbbVie, Amgen, Autolus, AvenCell, CanCell Therapeutics, Genmab US, Ichnos Sciences, Incyte Biosciences, Interius BioTherapeutics, Miltenyi Biomedicine, Molecular Partners, Nektar Therapeutics, Orbital Therapeutics, Pfizer. <b>C. Klein:</b> A. Employment (full or part-time); Significant; F. Hoffmann-La Roche Ltd. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; F. Hoffmann-La Roche Ltd. <b>S. Kobold:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; CR2 Inc., Tabby Therapeutics, Catalym GmBH, Plectonic GmBH, Arcus Bioscience. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; CR2 Inc., Miltenyi, Galapagos, Novartis, BMS, GS. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; CR2 Inc., Carina Biotech.